Activator-blocker model of transcriptional regulation by pioneer-like factors

Zygotic genome activation (ZGA) in the development of flies, fish, frogs and mammals depends on pioneer-like transcription factors (TFs). Those TFs create open chromatin regions, promote histone acetylation on enhancers, and activate transcription. Here, we use the panel of single, double and triple mutants for zebrafish genome activators Pou5f3, Sox19b and Nanog, multi-omics and mathematical modeling to investigate the combinatorial mechanisms of genome activation. We show that Pou5f3 and Nanog act differently on synergistic and antagonistic enhancer types. Pou5f3 and Nanog both bind as pioneer-like TFs on synergistic enhancers, promote histone acetylation and activate transcription. Antagonistic enhancers are activated by binding of one of these factors. The other TF binds as non-pioneer-like TF, competes with the activator and blocks all its effects, partially or completely. This activator-blocker mechanism mutually restricts widespread transcriptional activation by Pou5f3 and Nanog and prevents premature expression of late developmental regulators in the early embryo.

Regarding the mathematical modelling; in Figure 5c, expressions of 3b. P-N+ genes do not seem to be upregulated in MZps. They even seems down regulated at stages before 6 hpf. Furthermore, gene ontology analyses are not enough to evaluate the model accuracy, and validation in other method is required. For example, is there any differences in sequence feature between enhancers of the six groups sorted by the model?
Reviewer #2 (Remarks to the Author): The manuscript "Activator-blocker model of transcriptional regulation by pioneer-like factors," by Riesle et al takes a deep dive into functions for the transcription factors Pou5f3, Sox19b and Nanog during zebrafish zygotic genome activation. Their analysis uses a combination of elegant genetic and genomic approaches. The results reveal surprising complexities in the ways in which Pou5f3, Sox19b and Nanog work both together and antagonistically to regulate early transcription.
Overall this is a rigorous study which produces useful data sets and shifts the way we need to think about how these transcription factors act during zebrafish ZGA. I have only minor comments.
The authors do a very nice job of detailing the crosses performed to generate the various mutant lines in the methods, and the original alleles are cited. However, given the importance of the mutants it might be worth reviewing the nature of the alleles in the start of the results section. For example, do any of them produce proteins that could still have some DNA binding capacity? With respect to figure 2, it could be more helpful to include a little more detail on the binning of up down and unchanged groups in the text or figure legend (what were the cutoffs/criteria for including). I similarly struggled to find this information in the methods, if it is there it is not easy to find. With respect to figure 3 F, it isn't entirely clear to me why this group couldnt reflect a requirement for all three TFs together, rather than any one plus GC binding factor-Fig 3b seems to suggests binding motifs for all three are detected in this group, although the sox:pou motif is underrepresented. The GC enrichment could still represent a hypothetical GC binding factor required in addition to the three factors? Alternatively, is it possible that there is simply a structural property of these GC rich regions that is keeping them more open? I believe there is reasonable data to support a relationship between GC content and nucleosome occupancy  I think a prediction of the model in 4f is that you should never be able to IP A and B from chromatin together at these types of sites. Is it possible to do sequential IPs to explore that possibility? This could be interesting additional support, but if this is technically too challenging, could be viewed as beyond the current paper's scope.

Dear reviewers #1 and #2,
We are grateful that you professionally spotted the weak points in our manuscript, which helped us to improve it. We performed additional experiments, added new sub-chapter in the results section with new main Fig. 5 and Fig.S6, performed additional analysis and made changes in most of the figures. We numbered your comments and linked them as comments to the yellow-marked text in the PDF file "Riesle_main_and SUPPL_marked for reviewers" , which contains main text and figures, supplementary figures and legends and also additional supplementary Figure for Reviewer 1, at the end of the supplementary material. Please find detailed answers to your criticizms below. Sincerely, the authors.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this manuscript, H3K27ac ChIP-seq, and transcriptome of zebrafish mutants of ZGA regulator TFs. They found the existence of synergistic and antagonistic enhancer types, and Pou5f3 and Nanog can function as activator or blocker in antagonistic enhancers. The topic is unquestionably important for the field, but this reviewer feels that the novelty of the study seems weak. I have several major concerns as described below.
R1-1.Regarding the novelty, the fact that the pioneer factors (Pou5f3 and Sox19b) is preventing premature expression for some of the genes during ZGA has already been reported by the same group (Gao et al., 2022). The idea that Pou5f3 and Nanog is competing at a same binding site and may function as a blocker is indeed interesting and advancing our knowledge, but molecular experimental evidence is lacking. How Pou5f3 and Nanog function as a non-pioneer blocker is also completely a blackbox. To address underlying molecular mechanisms, at least the authors should do experiments such as gel-shift assay to enhancer sequences of specific group of genes.
A 1-1. We agree with the comment and provide requested evidence. We performed series of the gel-shift assays with the oligos from different enhancers, added the new figures 5 and S6 and a chapter "Pou5f3 and Nanog bind to the common binding sites in a mutually exclusive way". We also show that in most cases TdARs have only one match to either Pou5f3 or Nanog motif (new panel Fig. S5f). Fig. 2a, why did not the authors use the MZsox19b for the classification? Second, the results show that ~50% or ~80% of group 4.-TdARs can be rescued by Sox19b alone, or by Sox19b and Nanog, respectively (Fig S3). From these data, it is likely that SoxB, or SoxB and Nanog is required on chromatin accessibility of most of group 4.-TdARs.

R1-2. The authors claim the presence of additional genome activators, but the evidence is not enough. First, in
A1-2. We agree with both points. In response to this criticism we -made additional analysis of 4.-group, using ATAC-seq in MZsox19b and three double mutants (new panel c in the Fig. S3).
removed "hypothetical GC protein" from the scheme in Fig.3; and speculations about "hypothetical GC protein" from the corresponding text in the results.

R1-4. Furthermore, they mention that exact match to the motif is important for Pou5f3 and Nanog to function as pioneer TFs, but this does not explain the difference between synergistic and antagonistic enhancers. Why do Pou5f3 and Nanog function as blocker only in antagonistic enhancers, but not in synergistic enhancers?
The author also need to have additional experimental data as stated above.
A1-4. There are two parts of the answer to this criticizm: 1) Yes, we provided additional experimental data and confirmed now with gel-shifts, that the factor which binds stronger in-vitro works as an activator in-vivo (9 out of 9 oligos with single motif, where the binding worked for any of the two TFs, see the new Fig. 5 panel e). We also show that GC content is important: Pou5f3 activates the enhancers with lower GC content range, than Nanog, as judged by pioneer activity ( Fig.3e) and H3K27ac change (Fig.S4).
2) No, we can not explain the mechanistic difference between synergistic and antagonistic enhancers from gel-shifts and bulk genomic data. We assume that cell-specific cofactors are involved ( see discussion).
Single-cell analysis is required to answer this question; as far as we know ChIP-seq technique for single cells is not yet developed. We hope that our manuscript is novel enough to be published without it.. . Figure 5c, expressions of 3b. P-N+ genes do not seem to be upregulated in MZps. They even seems down regulated at stages before 6 hpf.f This is because most of 3b. P-N+ genes are coactivated by SOXB1 sum, and SOXB1 sum is decreased in MZps.( see the supplementary Figure for Reviewer 1 included in the file for reviewers). 3b. P-N+ model group consists of three mini-models: of three mini-models: S+P-N+ (best fit to 398 transcripts), S0P-N+ ( best fit to 24 transcripts) and S-P-N+ (best fit to 6 transcripts). In response to this criticism we changed the sumentary figure S8, which shows now the example fits to all minimodels ( and not to the groups as before).

R1-5. Regarding the mathematical modelling; in
R1-6. Furthermore, gene ontology analyses are not enough to evaluate the model accuracy, and validation in other method is required.
We did not intent to validate the modeling with GO analysis. In response to this criticizm we completly removed this GO analysis from the paper, not to distract the reader attention. We renamed the sub-chapters and put the cross-validation part (the synergistically and antagonistically regulated transcripts are linked to synergistically and antagonistically regulated enhancers) just after the description of the modeling results (see the marking of the text for reviewers).

R1-7.For example, is there any differences in sequence feature between enhancers of the six groups sorted by the model?
Yes, there is a difference in both sequence features: motif frequency and in GC content. We show it now in Fig.  S9 c,d, as additional validation.
Reviewer #2 (Remarks to the Author): The manuscript "Activator-blocker model of transcriptional regulation by pioneer-like factors," by Riesle et al takes a deep dive into functions for the transcription factors Pou5f3, Sox19b and Nanog during zebrafish zygotic genome activation. Their analysis uses a combination of elegant genetic and genomic approaches. The results reveal surprising complexities in the ways in which Pou5f3, Sox19b and Nanog work both together and antagonistically to regulate early transcription.
Overall this is a rigorous study which produces useful data sets and shifts the way we need to think about how these transcription factors act during zebrafish ZGA. I have only minor comments.
R2-1. The authors do a very nice job of detailing the crosses performed to generate the various mutant lines in the methods, and the original alleles are cited. However, given the importance of the mutants it might be worth reviewing the nature of the alleles in the start of the results section. For example, do any of them produce proteins that could still have some DNA binding capacity?
A2-1. All the mutants are genetic nulls: Pou5f3 mutant has a point-mutation before DNA-binding domain, Sox19b and Nanog have TALEN-induced frameshifts before the DNA-binding domains. We mention it in the results and give the references to the original publications where it was characterized after each mutant name.